Sphingolipids (SLs) are essential structural components of membranes and provide reservoirs for critical bioactive lipids, including ceramide and sphingosine 1-phosphate (S1P). Whereas ceramide is associated with apoptosis and growth arrest, S1P commonly promotes survival and proliferation. The best- characterized targets of S1P are S1P receptors localized to the plasma membrane. S1P also can act in the nucleus by binding to and inhibiting class I histone deacetylases (HDACs), which are major regulators of gene expression. SL signaling is characterized by highly compartmentalized generation and targeting of the bioactive molecules. Substantial insight on localized S1P production and targeting at the plasma membrane has been obtained, but little is known about regulation of SL metabolism in the nucleus. This project involves investigation of proteins with potential roles in generating bioactive SLs in the nucleus. The work includes analysis of a neutral sphingomyelinase localized to the nuclear envelope (NE) that is required for myoblast differentiation, which may be integral to nuclear SL metabolism. This component and other enzymes with potential links to nuclear SL metabolism will be analyzed in a cultured myoblast differentiation model. Subcellular fractionation and lipidomics will be used to characterize the role of these proteins in regulating the dynamic SL landscape of the nucleus during myoblast differentiation, and in producing bioactive SLs including S1P. In addition, the potential functions of S1P in activating expression of myogenic genes as an inhibitory ligand for class I HDACs will be evaluated. Overall this project will investigate the hypothesis that the nucleus houses a distinctive SL metabolism that has a major role in myogenic differentiation. The work has clear relevance to muscle pathology in humans, since HDAC inhibitors including S1P have been shown to ameliorate muscular dystrophies in animal models.